ELISA kit for detecting ractopamine residue and method of use thereof

An enzyme-linked immunosorbent reagent, ractopamine technology, applied in the field of enzyme-linked immunosorbent immunoassay kits for the detection of ractopamine residues in animal-derived foods, can solve the problems of tediousness, high equipment requirements, and many steps

Active Publication Date: 2008-08-13
SOUTH CHINA AGRI UNIV
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  • Abstract
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Problems solved by technology

[0009] In addition, searching related patents found that the Chinese patent "An ELISA Kit for Detecting Ractopamine in Foods of Animal Origin" (Application No. 200510086776.0) mentioned an ELISA detection method and kit for ractopamine. The enzyme-linked immunoassay method mentioned in is that the ELISA plate is coated with ractopamine antigen, and the sample to be tested is added to react with ractopamine monoclonal antibody or polyclonal antibody, the plate is washed, and then enzyme-labeled anti-antibody reaction is added. After washing, Add substrate for color development, after termination, read; or if the ELISA plate is coated with anti-antibody, first add ractopamine monoclonal antibody or polyclonal antibody to react, wash the plate, then add the sample to compete with the enzyme-labeled ractopamine antigen for reaction, wash After that, add the substrate to develop the color, and after the termination, take the reading; both methods are indirect competitive ELISA, the operation is complicated, the steps are many, the production and storage costs are high, and it is difficult to meet the needs of practical applications
[0010] The Chinese patent "Affinity Chromatography-ELISA Method for Ractopamine and Its Special Kit" (Application No. 200510071059.0) mentions a ractopamine ELISA method and kit. The enzyme mentioned in this patent In the immunoassay method, the sample is firstly purified by immunoaffinity chromatography, then added to the microplate plate coated with ractopamine antibody for reaction, after washing, the enzyme-labeled antigen is added for reaction, after washing, color development, termination, and reading; In this method, the sample needs to be purified and enriched through an affinity chromatography column at first. The operation is complicated and there are many steps, and the detection mode is indirect competition ELISA. Meet the needs of actual large-throughput detection
[0011] In short, the existing enzyme-linked immunoassay methods for ractopamine at home and abroad are complicated and cumbersome, and are difficult to apply in practice, and the existing products are generally poor in stability, complicated in sample pretreatment and detection steps, high in equipment requirements, and expensive. Expensive and other deficiencies have seriously affected the detection and monitoring of ractopamine residues. Therefore, it is of great economic and social significance to develop a ractopamine ELISA kit with high stability, simple operation, low equipment requirements, and low cost.

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  • ELISA kit for detecting ractopamine residue and method of use thereof

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Embodiment 1

[0085] The preparation of embodiment 1 antigen

[0086] Preparation of ractopamine antigen:

[0087] a. Dissolve 50 μmol / L ractopamine hapten in 1 mL of DMF, then add equimolar DCC and NHS to the solution, and let it react overnight at room temperature;

[0088] b. Centrifuge, take 800 μL of supernatant, slowly add to 4 mL of 15 mg / mL BSA or OVA carrier protein carbonic acid buffer solution, and then react for 4 hours under magnetic stirring;

[0089] c. After the reaction is completed, put it into a dialysis bag, first dialyze twice with distilled water, and then dialyze with 0.8% normal saline to obtain the product;

[0090] d. The binding ratio is determined by ultraviolet scanning, and finally the antigen is concentrated and stored or freeze-dried to obtain the ractopamine immunogen and the coating source, which are divided and stored in a refrigerator at -20°C.

Embodiment 2

[0091] The preparation of embodiment 2 antibody

[0092] Preparation of ractopamine mouse monoclonal antibody:

[0093] Animal immunization procedure: Balb / c mice were used as immunized animals, the ractopamine hapten and bovine serum albumin conjugate was used as the immunogen, the immunization dose was 60 μg / mouse, and the immunogen was mixed with the same amount of Freund’s The complete adjuvant was mixed to make an emulsifier, which was injected intraperitoneally. The same dose of immunogen and the same amount of Freund's incomplete adjuvant were mixed and emulsified at intervals of 3 weeks, and a booster immunization was given.

[0094] Cell fusion and cloning: Splenocytes from immunized Balb / c mice were fused with SP2 / 0 myeloma cells at a ratio of 4:1. Cell supernatants were measured by indirect competitive enzyme-linked immunosorbent assay, and positive wells were screened. The positive wells were cloned by microcloning until a hybridoma cell line stably secreting the ...

Embodiment 3

[0097] The extraction of embodiment 3 horseradish peroxidase or alkaline phosphatase

[0098] 1. Extraction of horseradish peroxidase

[0099] a. Water extraction: take by weighing 20 kilograms of washed fresh horseradish or horseradish skin, cut into small pieces, and mince in a pulverizer. Add 10 kg of water to the crushed slag slurry, stir and extract at low temperature for 8 hours, centrifuge at 3000 rpm for 10 minutes, and collect the supernatant.

[0100] b. Fractional separation of ammonium sulfate: add 226 grams of ammonium sulfate powder per liter of filtrate, stir while adding, put overnight at room temperature. The next day, draw the supernatant, and then add 258 grams of ammonium sulfate powder per liter of supernatant, and stir as you add it. After the ammonium sulfate is completely dissolved, put it in a cold room overnight. The next day, the supernatant was sucked off, and the precipitated part was centrifuged at 13,000 rpm for 20 minutes in a refrigerated cen...

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Abstract

The present invention discloses enzyme-linked immune kits for detecting ractopamine including antigenic enzyme label plate coated with ractopamine, enzyme labeled ractopamine antibody working fluid, standard ractopamine solution, substrate solution, substrate buffer solution, reaction termination liquid, condense washing liquid and sample diluted concentrated liquid. The present invention also discloses using method for detecting ractopamine residue by said kits including steps of pretreatment of sample, detecting with kits, result process and analysis and so on. The kits provided by present invention employ directly competing enzyme-linked immunoadsorption analysis has merits of high sensitivity and good stability, simplify greatly the operation steps and reaction time to reduce error brought by complex operation, reduce cost and suit for screening of a large mount of samples, and has importance practical significance.

Description

technical field [0001] The invention relates to the technical field of ELISA detection, in particular to an ELISA kit for detecting ractopamine residues in animal-derived foods and a method for using the same. Background technique [0002] Ractopamine is a kind of β2-adrenergic agonist (BAA), and it belongs to catecholamines (adrenaline and norepinephrine) substances like "leptbuterol". As early as the early 1970s, the ability of catecholamines and their analogs to improve the nutritional allocation and carcass composition of the body was recognized by the research department of the American cyanamide company. Then it is used on a large scale in livestock and poultry production to increase lean meat percentage, reduce fat deposition, and increase milk production. Among them, ractopamine and clenbuterol are the most widely used in production because of their high oral efficiency. [0003] In recent years, ractopamine and clenbuterol have been added to feed in large quantiti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/535G01N33/577
Inventor 孙远明杨金易雷红涛沈玉栋肖治理王弘张挺
Owner SOUTH CHINA AGRI UNIV
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